The Raf protein serine/threonine kinase is a crucial mediator of mitogenic signal transduction, and its altered expression or activity has been associated with several types of human cancer, including lung. We developed a mouse model for induction of this tumor type and examined it for Raf involvement. Tumors were inducted by transplacental injection of 1-ethyl-1-nitrosourea followed by promotion with butylated hydroxytoluene. This regimen induces lung adenocarcinomas in 100% of offspring and T-cell lymphomas in 70%. Examination of tumors for altered expression or structure of a panel of oncogenes revealed consistent mutations of c-raf-1, along with a conspicuous lack of Ras mutations. All of these mutations result in amino acid substitutions, and functional assays for mutated Raf-1 demonstrated that the mutations were activating. This is the first example of point-mutated c-raf-1 in vivo, demonstrating a new class of activating raf mutations. Their location in subdomain VIII of the kinase domain at the surface of the substrate pocket suggests that they might be exploited for the development of mutant-specific inhibitors.